SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model.

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.

Co-delivery of a trimeric spike DNA and protein vaccine with aluminum hydroxide enhanced Th1-dominant humoral and cellular immunity against SARS-CoV-2.

Protein subunit vaccines have been used as prophylactic vaccines for a long time. The well-established properties of these vaccines make them the first choice for the coronavirus disease 2019 (COVID-19) outbreak. However, it is not easy to develop a protein vaccine that induces cytotoxic T lymphocyte responses and requires a longer time for manufacturing, which limits the usage of this vaccine type. Here, we report the combination of a recombinant spike (S)-trimer protein with a DNA vaccine-encoded S protein as a novel COVID-19 vaccine. The recombinant S protein was formulated with different adjuvants and mixed with the DNA plasmid before injection. We found that the recombinant S protein formulated with the adjuvant aluminum hydroxide and mixed with the DNA plasmid could enhance antigen-specific antibody titers, neutralizing antibody titers. We further evaluated the IgG2a/IgG1 isotype and cytokine profiles of the specific boosted T-cell response, which indicated that the combined vaccine induced a T-helper 1 cell-biased immune response. Immunized hamsters were challenged with severe acute respiratory syndrome coronavirus 2, and the body weight of the hamsters that received the recombinant S protein with aluminum hydroxide and/or the DNA plasmid was not reduced. Alternatively, those that received control or only the DNA plasmid immunization were reduced. Interestingly, after the third day of the viral load in the lungs, the viral challenge could not be detected in hamsters immunized with the recombinant S protein in aluminum hydroxide mixed with DNA (tissue culture infectious dose < 10). The viral load in the lungs was 109 , 106 , and 107 for the phosphate-buffered saline, protein in aluminum hydroxide, and DNA-only immunizations, respectively. These results indicated that antiviral mechanisms neutralizing antibodies play important roles. Furthermore, we found that the combination of protein and DNA vaccination could induce relatively strong CD8+ T-cell responses. In summary, the protein subunit vaccine combined with a DNA vaccine could induce strong CD8+ T-cell responses to increase antiviral immunity for disease control.

Omicron-specific mRNA vaccine induced cross-protective immunity against ancestral SARS-CoV-2 infection with low neutralizing antibodies.

The major challenge in COVID-19 vaccine effectiveness is immune escape by SARS-CoV-2 variants. To overcome this, an Omicron-specific messenger RNA (mRNA) vaccine was designed. The extracellular domain of the spike of the Omicron variant was fused with a modified GCN4 trimerization domain with low immunogenicity (TSomi). After immunization with TSomi mRNA in hamsters, animals were challenged with SARS-CoV-2 virus. The raised nonneutralizing antibodies or cytokine secretion responses can recognize both Wuhan S and Omicron S. However, the raised antibodies neutralized SARS-CoV-2 Omicron virus infection but failed to generate Wuhan virus neutralizing antibodies. Surprisingly, TSomi mRNA immunization protected animals from Wuhan virus challenge. These data indicated that non-neutralizing antibodies or cellular immunity may play a more important role in vaccine-induced protection than previously believed. Next-generation COVID-19 vaccines using the Omicron S antigen may provide sufficient protection against ancestral or current SARS-CoV-2 variants.

Neutralizing antibodies targeting a novel epitope on envelope protein exhibited broad protection against flavivirus without risk of disease enhancement.

BACKGROUND: Flavivirus causes many serious public health problems worldwide. However, licensed DENV vaccine has restrictions on its use, and there is currently no approved ZIKV vaccine. Development of a potent and safe flavivirus vaccine is urgently needed. As a previous study revealed the epitope, RCPTQGE, located on the bc loop in the E protein domain II of DENV, in this study, we rationally designed and synthesized a series of peptides based on the sequence of JEV epitope RCPTTGE and DENV/ZIKV epitope RCPTQGE. METHODS: Immune sera were generated by immunization with the peptides which were synthesized by using five copies of RCPTTGE or RCPTQGE and named as JEV-NTE and DV/ZV-NTE. Immunogenicity and neutralizing abilities of JEV-NTE or DV/ZV-NTE-immune sera against flavivirus were evaluated by ELISA and neutralization tests, respectively. Protective efficacy in vivo were determined by passive transfer the immune sera into JEV-infected ICR or DENV- and ZIKV-challenged AG129 mice. In vitro and in vivo ADE assays were used to examine whether JEV-NTE or DV/ZV-NTE-immune sera would induce ADE. RESULTS: Passive immunization with JEV-NTE-immunized sera or DV/ZV-NTE-immunized sera could increase the survival rate or prolong the survival time in JEV-challenged ICR mice and reduce the viremia levels significantly in DENV- or ZIKV-infected AG129 mice. Furthermore, neither JEV -NTE- nor DV/ZV-NTE-immune sera induced antibody-dependent enhancement (ADE) as compared with the control mAb 4G2 both in vitro and in vivo. CONCLUSIONS: We showed for the first time that novel bc loop epitope RCPTQGE located on the amino acids 73 to 79 of DENV/ZIKV E protein could elicit cross-neutralizing antibodies and reduced the viremia level in DENV- and ZIKV-challenged AG129 mice. Our results highlighted that the bc loop epitope could be a promising target for flavivirus vaccine development.

Induction of high affinity monoclonal antibodies against SARS-CoV-2 variant infection using a DNA prime-protein boost strategy.

BACKGROUND: Calls for the coronavirus to be treated as an endemic illness, such as the flu, are increasing. After achieving high coverage of COVID-19 vaccination, therapeutic drugs have become important for future SARS-CoV-2 variant outbreaks. Although many monoclonal antibodies have been approved for emergency use as treatments for SARS-CoV-2 infection, some monoclonal antibodies are not authorized for variant treatment. Broad-spectrum monoclonal antibodies are unmet medical needs. METHODS: We used a DNA prime-protein boost approach to generate high-quality monoclonal antibodies. A standard ELISA was employed for the primary screen, and spike protein-human angiotensin-converting enzyme 2 blocking assays were used for the secondary screen. The top 5 blocking clones were selected for further characterization, including binding ability, neutralization potency, and epitope mapping. The therapeutic effects of the best monoclonal antibody against SARS-CoV-2 infection were evaluated in a hamster infection model. RESULTS: Several monoclonal antibodies were selected that neutralize different SARS-CoV-2 variants of concern (VOCs). These VOCs include Alpha, Beta, Gamma, Delta, Kappa and Lambda variants. The high neutralizing antibody titers against the Beta variant would be important to treat Beta-like variants. Among these monoclonal antibodies, mAb-S5 displays the best potency in terms of binding affinity and neutralizing capacity. Importantly, mAb-S5 protects animals from SARS-CoV-2 challenge, including the Wuhan strain, D614G, Alpha and Delta variants, although mAb-S5 exhibits decreased neutralization potency against the Delta variant. Furthermore, the identified neutralizing epitopes of monoclonal antibodies are all located in the receptor-binding domain (RBD) of the spike protein but in different regions. CONCLUSIONS: Our approach generates high-potency monoclonal antibodies against a broad spectrum of VOCs. Multiple monoclonal antibody combinations may be the best strategy to treat future SARS-CoV-2 variant outbreaks.

Low-Dose SARS-CoV-2 S-Trimer with an Emulsion Adjuvant Induced Th1-Biased Protective Immunity.

During the sustained COVID-19 pandemic, global mass vaccination to achieve herd immunity can prevent further viral spread and mutation. A protein subunit vaccine that is safe, effective, stable, has few storage restrictions, and involves a liable manufacturing process would be advantageous to distribute around the world. Here, we designed and produced a recombinant spike (S)-Trimer that is maintained in a prefusion state and exhibits a high ACE2 binding affinity. Rodents received different doses of S-Trimer (0.5, 5, or 20 µg) antigen formulated with aluminum hydroxide (Alum) or an emulsion-type adjuvant (SWE), or no adjuvant. After two vaccinations, the antibody response, T-cell responses, and number of follicular helper T-cells (Tfh) or germinal center (GC) B cells were assessed in mice; the protective efficacy was evaluated on a Syrian hamster infection model. The mouse studies demonstrated that adjuvating the S-Trimer with SWE induced a potent humoral immune response and Th1-biased cellular immune responses (in low dose) that were superior to those induced by Alum. In the Syrian hamster studies, when S-Trimer was adjuvanted with SWE, higher levels of neutralizing antibodies were induced against live SARS-CoV-2 from the original lineage and against the emergence of variants (Beta or Delta) with a slightly decreased potency. In addition, the SWE adjuvant demonstrated a dose-sparing effect; thus, a lower dose of S-Trimer as an antigen (0.5 µg) can induce comparable antisera and provide complete protection from viral infection. These data support the utility of SWE as an adjuvant to enhance the immunogenicity of the S-Trimer vaccine, which is feasible for further clinical testing.

Recombinant lipidated Zika virus envelope protein domain III elicits durable neutralizing antibody responses against Zika virus in mice.

BACKGROUND: The emergence of Zika virus (ZV) in tropical and subtropical areas of the world has created an urgent need for vaccines against ZV. However, approved vaccines that prevent ZV infection are not available. To develop an effective vaccine against ZV infection, a lipidated form of ZV envelope protein domain III that possesses an intrinsic adjuvant property was rationally designed. Our goal was to examine the immunogenicity of recombinant lipidated ZV envelope protein domain III (rLZE3) and evaluate its potential as a vaccine candidate against ZV. METHODS: Recombinant ZV envelope protein domain III (rZE3) and rLZE3 were prepared with an Escherichia coli-based system. Dendritic cell surface marker expression and cytokine production upon stimulation were analyzed to evaluate the function of rLZE3. Neutralizing antibody capacities were evaluated using focus reduction neutralization tests after immunization. To investigate the protective immunity in immunized mice, serum samples collected from immunized mice were adoptively transferred into AG129 mice, and then viremia levels and survival times were examined after ZV challenge. RESULTS: rLZE3 alone but not rZE3 alone efficiently activated dendritic cells in vitro and was taken up by dendritic cells in vivo. Immunization of C57BL/6 mice with rLZE3 alone (without exogenous adjuvant) could induce ZV-specific neutralizing antibody responses. Furthermore, serum samples obtained from rLZE3-immunized mice provided protection as indicated by a reduction in viremia levels and prolongation of survival times after ZV challenge. CONCLUSION: These results indicate that rLZE3 is an excellent vaccine candidate and has great potential that should be evaluated in further preclinical studies.

Domain 4 of pneumolysin from Streptococcus pneumoniae is a multifunctional domain contributing TLR4 activating and hemolytic activity.

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.

Molecular mechanisms of TLR2-mediated antigen cross-presentation in dendritic cells.

Cross-presentation is a key function of dendritic cells (DCs), which present exogenous Ags on MHC class I molecules to prime CTL responses. The effects of TLR triggering on the cross-presentation of exogenous Ags by DCs remain unclear. In this study, we used synthetic dipalmitoylated peptides and TLR2 agonist-conjugated peptides as models to elucidate the mechanisms of TLR2-mediated cross-presentation. We observed that the internalization of dipalmitoylated peptides by bone marrow-derived DCs was facilitated by TLR2 via clathrin-mediated endocytosis. The administration of these dipalmitoylated peptide-pulsed bone marrow-derived DCs eliminated established tumors through TLR2 signaling. We further demonstrated that the induction of Ag-specific CTL responses and tumor regression by dipalmitoylated peptides was TAP independent. In addition, presentation of dipalmitoylated peptides by MHC class I molecules was blocked in the presence of an endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO). The endocytosed dipalmitoylated peptide also passed rapidly from early endosome Ag-1-positive endosomes to RAS-related GTP-binding protein 7 (Rab7)-associated late endosomes compared with their nonlipidated counterparts. Furthermore, we found that dipalmitoylated peptide-upregulated Rab7 expression correlated with Ag presentation via the TLR2/MyD88 pathway. Both JNK and ERK signaling pathways are required for upregulation of Rab7. In summary, our data suggest that TLR2-mediated cross-presentation occurs through the upregulation of Rab7 and a TAP-independent pathway that prime CTL responses.

Recombinant lipoprotein-based vaccine candidates against C. difficile infections.

BACKGROUND: Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli. RESULTS: The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 µg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 µg of TcdA toxoid or 30 µg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin. CONCLUSION: These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.

Mesenchymal stem cells tune the development of monocyte-derived dendritic cells toward a myeloid-derived suppressive phenotype through growth-regulated oncogene chemokines.

Mesenchymal stem/stromal cells (MSCs) are promising potential candidates for the treatment of immunological diseases because of their immunosuppressive functions. However, the molecular mechanisms that mediate MSCs' immunosuppressive activity remain elusive. In this article, we report for the first time, to our knowledge, that secreted growth-regulated oncogene (GRO) chemokines, specifically GRO-γ, in human MSC-conditioned media have an effect on the differentiation and the function of human monocyte-derived dendritic cells. The monocyte-derived dendritic cells were driven toward a myeloid-derived suppressor cell (MDSC)-like phenotype by the GRO chemokines. GRO-γ-treated MDSCs had a tolerogenic phenotype that was characterized by an increase in the secretion of IL-10 and IL-4, and a reduction in the production of IL-12 and IFN-γ. We have also shown that the mRNA expression levels of the arginase-1 and inducible NO synthase genes, which characterize MDSCs, were upregulated by GRO-γ-primed mouse bone marrow cells. In addition, the ability of GRO-γ-treated bone marrow-derived dendritic cells to stimulate the OVA-specific CD8(+) T (OT-1) cell proliferation and the cytokine production of IFN-γ and TNF-α were significantly decreased in vivo. Our findings allow a greater understanding of how MDSCs can be generated and offer new perspectives to exploit the potential of MDSCs for alternative approaches to treat chronic inflammation and autoimmunity, as well as for the prevention of transplant rejection.

Toll-like receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated immunosuppressive cells numbers in a mouse cervical cancer model following recombinant lipoprotein therapy.

BACKGROUND: Although cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors. METHODS: Mouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry. RESULTS: We demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. CONCLUSION: These findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional therapeutic cancer vaccines.

A poorly neutralizing IgG2a/c response elicited by a DNA vaccine protects mice against Japanese encephalitis virus.

We demonstrated previously that immunization with a DNA vaccine expressing the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection through a poorly neutralizing antibody response. Here, we further investigated the role of the IgG subclass in this antibody-dependent protection using cytokine co-immunization and cytokine-deficient mice. A significant difference in IgG2a/c but not IgG1 was observed between mice that survived or died following a lethal challenge. Correspondingly, the IgG2a/c response and protection increased in IL-4-deficient mice but decreased in IFN-γ-deficient mice, highlighting the importance of IgG2a/c. In addition, the restoration of protection and E-specific IgG2a/c production in IFN-γ-deficient mice by a T helper (Th) type 1-biased intramuscular immunization suggested that IgG2a/c but not IFN-γ was the major component for protection. The failure of protection against a direct intracranial challenge indicated that IgG2a/c-mediated protection was restricted to outside the central nervous system. Consistent with this conclusion, passive transfer of E-specific antisera conferred protection only pre-exposure to JEV. Therefore, our data provided evidence that the IgG subclass plays an important role in protection against JEV, particular in poorly neutralizing E-specific antibodies, and Th1-biased IgG2a/c confers better protection than Th2-biased IgG1 against JEV.

CD4+ T cells disarm or delete cytotoxic T lymphocytes under IL-17-polarizing conditions.

Previous studies have shown that TGF-ß acts cooperatively with IL-6 to elicit a high frequency of IL-17-secreting CD4(+) T cells (termed Th17) and an elevated CD8(+)IL-17(+) T cell population (termed Tc17). These CD8(+) cells fail to behave like most cytotoxic T lymphocytes that express IFN-γ and granzyme B, but they exhibit a noncytotoxic phenotype. Although a significant increase in the number of these Tc17 cells was found in tumors, their role and interaction with other cell types remain unclear. In this study, we demonstrate that the presence of CD4(+)CD25(-) T cells, but not the CD4(+)CD25(+) (regulatory T [Treg]) cell population, significantly reduced the elicitation of Tc17 cells, possibly as a result of the induction of apoptotic signals. Importantly, these signals may be derived from soluble mediators, and the addition of anti-IL-2 restored the reduction of Tc17 cells in the presence of CD4(+)CD25(-) T cells. Finally, the elicited Tc17 and Treg cells exhibited a close association in patients with head and neck cancer, indicating that the surrounding Treg cells might maintain the survival of the Tc17 cells. Taken together, these results reveal an intriguing mechanism in which Tc17 cells are controlled by a finely tuned collaboration between the different types of CD4(+) T cells in distinct tumor microenvironments.

Enhancing Cancer Therapy: Boron-Rich Polyboronate Ester Micelles for Synergistic Boron Neutron Capture Therapy and PD-1/PD-L1 Checkpoint Blockade.

Malignant cancers, known for their pronounced heterogeneity, pose substantial challenges to monotherapeutic strategies and contribute to the risk of metastasis. Addressing this, our study explores the synergistic potential of combining boron neutron capture therapy (BNCT) with immune checkpoint blockade to enhance cancer treatment efficacy. We synthesized boron-rich block copolymer micelles as a novel boron drug for BNCT. Characterization was conducted using nuclear magnetic resonance, gel-permeation chromatography, transmission electron microscopy, and dynamic light scattering. These micelles, with an optimal size of 91.3 nm and a polydispersity index of 0.18, are suitable for drug delivery applications. In vitro assessments on B16-F10 melanoma cells showed a 13-fold increase in boron uptake with the micelles compared to borophenyl alanine (BPA), the conventional boron drug for BNCT. This resulted in a substantial increase in BNCT efficacy, reducing cell viability to 77% post-irradiation in micelle-treated cells, in contrast to 90% in BPA-treated cells. In vivo, melanoma-bearing mice treated with these micelles exhibited an 8-fold increase in boron accumulation in tumor tissues versus those treated with BPA, leading to prolonged tumor growth delay (5.4 days with micelles versus 3.3 days with BPA). Moreover, combining BNCT with anti-PD-L1 immunotherapy further extended the tumor growth delay to 6.6 days, and enhanced T-cell infiltration and activation at tumor sites, thereby indicating a boosted immune response. This combination demonstrates a promising approach by enhancing cytotoxic T-cell priming and mitigating the immunosuppressive effects of melanoma tumors.

Effect of C-type lectin 16 on dengue virus infection in Aedes aegypti salivary glands.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins and an important component of mosquito saliva. Although CTLs play key roles in immune activation and viral pathogenesis, little is known about their role in regulating dengue virus (DENV) infection and transmission. In this study, we established a homozygous CTL16 knockout Aedes aegypti mutant line using CRISPR/Cas9 to study the interaction between CTL16 and viruses in mosquito vectors. Furthermore, mouse experiments were conducted to confirm the transmission of DENV by CTL16-/- A. aegypti mutants. We found that CTL16 was mainly expressed in the medial lobe of the salivary glands (SGs) in female A. aegypti. CTL16 knockout increased DENV replication and accumulation in the SGs of female A. aegypti, suggesting that CTL16 plays an important role in DENV transmission. We also found a reduced expression of immunodeficiency and Janus kinase/signal transducer and activator of transcription pathway components correlated with increased DENV viral titer, infection rate, and transmission efficiency in the CTL16 mutant strain. The findings of this study provide insights not only for guiding future investigations on the influence of CTLs on immune responses in mosquitoes but also for developing novel mutants that can be used as vector control tools.

Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters.

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates.

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

Enhancing Spiritual Well-Being, Physical Activity, and Happiness in Hospitalized Older Adult Patients with Swallowing Difficulties: A Comparative Study of Thickeners and Swallowing Exercises.

Swallowing difficulties often occur in older adult patients during acute hospitalization, leading to reduced nutritional intake, increased frailty, and various psychosocial challenges. This randomized controlled study aimed to assess the effects of two interventions, thickeners and swallowing exercises, on the spiritual well-being, physical activity, and happiness of older adult patients with swallowing difficulties during acute hospitalization from October 2019 to August 2020. Sample size calculation was performed using a conservative estimate approach, resulting in an estimate-required sample size of 42 participants. The sampling method was a random cluster sampling approach, with three ward rooms assigned to the thickeners group, swallowing exercises group and control group, respectively. Seventy-two participants were assigned to the intervention groups (thickeners or swallowing exercises) or the control group using a 1:1:1 stratified random assignment. Data were collected before and after the intervention, and matched samples were analyzed using t-tests, ANOVA, and generalized estimating equations for statistical analysis. Both intervention groups showed significant improvements in spiritual well-being (p < 0.001), physical activity (p < 0.001), and happiness (p < 0.001) compared to the control group. However, there were no significant differences between the intervention groups. Our findings suggest that interventions involving thickeners and swallowing exercises have positive effects on the spiritual well-being, physical activity, and happiness of older adult patients with swallowing difficulties during acute hospitalization and emphasize the importance of implementing these interventions to enhance the overall well-being and quality of life of this vulnerable patient population.